Interpretive Summary: Reproductive loss during early pregnancy is one of the most costly factors influencing the livestock industry. A large majority of reproductive failure in sheep occur as a result of an impaired signal from the developing conceptus to the maternal system during implantation. In sheep, several genes controlling the conceptus signaling product [ovine interferon-tau (oIFNt), i.e., oIFNt-o10, oIFNt-o8, oIFNt-o2] are known, but molecular mechanisms of tissue-specific gene expression and down-regulation of the IFNt genes are not known. The objective of this study was to evaluate molecular mechanisms responsible for cell transcription by three oIFNt genes using transient transfection analyses. Results provide evidence that a 22-bp sequence of the proximal promotor region, including the Ets-2 binding site, of the most active oIFNt-o10 gene was required for the less active oIFNt-o8 and oIFNt-o2 genes to become equally active in transient transfection reporter assays. Thorough understanding of conceptus gene regulation should enable improvement in survival throughout gestation.

Technical Abstract:
In ruminant ungulates, a conceptus secretory protein, interferon-tau (IFNt), is known to be a major protein implicated in the process of maternal recognition of pregnancy. However, molecular mechanisms by which the IFNt gene is regulated in a temporal and spatial manner have not been fully elucidated. IFNt belongs to a gene family like other type I IFNs and more than 10 IFNt gene sequences have been characterized. Approximately 75% of ovine IFNt (oIFNt) expressed in utero is derived from one gene called oIFNt-o10 and amounts of transcripts from other oIFNt genes such as oIFNt-o8 or oIFNt-o2 are minimal. It was hypothesized that the variation in expression levels exhibited by oIFNt-o10 and oIFNt-o8/-o2 genes was due to differences in the 5'-upstream sequences, particularly the presence or absence of the Ets-2 binding site in the proximal promoter regions of these oIFNt genes. To test this hypothesis, transient transfection experiments with human choriocarcinoma JEG3 cells were executed with deleted and/or mutated 5'-upstream regions of these oIFNt genes attached to the chloramphenicol acetyltransferase (CAT) reporter gene. Because only the Ets-2 binding site located in the oIFNt-o10 gene appeared to differentiate the expression levels of these constructs, the 6 base pair (bp) Ets-2 sequence from the oIFNt-o10 gene inserted into the oIFNt-o8/-o2 gene reporter construct was examined. The insertion of this Ets-2 binding site into the oIFNt-o8/-o2 reporter construct failed to increase the degree of transactivation. Rather than this 6 bp sequence, a 22 bp sequence of the proximal promoter region, including the Ets-2 binding site, of the oIFNt-o10 gene was required for oIFNt-o8/-o2-reporter transactivation. By electrophoretic mobility shift assay (EMSA), nuclear protein(s) bound to this 22 bp from the oIFNt-o10 and oIFNt-o8/-o2 genes differed. These results suggest that the short promoter region including the Ets-2 binding site, not the Ets-2 binding region itself, may determine different levels of oIFNt gene expressions seen in utero.